Cyclic depsipeptides have numerous uses in pharmacology. As an example, the depsipeptides disclosed in WO2009/024527 are useful for treatment of various diseases. For example, the compound of formula II mentioned in WO2009/024527 is useful for the treatment and prevention of inflammatory and/or hyperproliferative and pruritic skin diseases such as atopic dermatitis, psoriasis, pustular psoriasis, rosacea, keloids, hypertrophic scars, acne, Netherton's syndrome or other pruritic dermatoses such as prurigo nodularis, unspecified itch of the elderly as well as other diseases with epithelial barrier dysfunction such as aged skin.
Nostopeptin BN920, formerly isolated from the cyanobacterium Nostoc, was isolated also from Microcystis. Nostopeptin BN920 inhibited chymotrypsin with an IC50 value of 31 nM (see J. Nat. Prod. 68(9), 1324-7 (2005)).
These compounds can be produced by fermentation (using chondromyces croactus, myxobacteria) along with other depsipeptides comprising the so-called ahp-substructure (ahp: 3-amino-6-hydroxy-piperidin-2-one) and the corresponding dehydro-ahp substructure (dehydro-ahp: 3-amino-3,4-dihydro-1H-pyridin-2-one), also called “dehydrate” herein, respectively. Therefore, the yield of fermentation with regard to any single of these compounds is rather low.
The present invention relates to processes or methods that allow obtaining such cyclic depsipeptides with increased yield and/or in good purity.
In view of the many risks, such as epimerization, tautomerization and the like in the synthesis of a complex molecule with many possible isomers, it has been possible to find a manufacturing process, preferably comprising a mixture of solid phase peptide synthesis and reactions in solution, that allows to produce cyclic depsipeptides of formula I in good yield and/or the required stereoisomerical purity, especially both. It is possible to reduce the amount of by-products, and even to improve yield, by converting such by-products, especially the dehydro-ahp substructure and/or an analogue of the desired ahp-comprising products with a five-membered ring instead of the ahp, into the desired final products. This allows to further increase yield. No synthesis has so far come to our attention making use of solid phase peptide synthesis in this field.    (i/a) In a first embodiment, the invention relates to a method or process for the preparation of a cyclic depsipeptide compound of the formula I,
    especially of the formula IA
    wherein    A1 is a bivalent moiety of an amino acid with a terminal carboxy or carbamoyl group, especially asparagine or glutamine, and is bound at its right hand side in formula I (corresponding to the C-terminus) via a carbonyl (preferably the carbonyl of an α-carboxyl group thereof) to the rest of the molecule; or is C1-8-alkanoyl or phosphorylated hydroxy-C1-8-alkanoyl;    X is bound via an N of A1 and is acyl, or is absent if A1 is C1-8-alkanoyl or phosphorylated hydroxy-C1-8-alkanoyl;    R2 is C1-8-alkyl, especially methyl;    R3 is the side chain of an amino acid, especially of leucine, isoleucine or valine;    R5 is the side chain of an amino acid, preferably of phenylalanine, leucine, isoleucine or valine;    R6 is the side chain of a hydroxy amino acid, especially of tyrosine;    R7 is the side chain of an amino acid, preferably of the amino acid leucine, isoleucine or valine; and    Y is hydrogen or C1-8-alkyl;    or a salt thereof,    said method comprising    selectively deprotecting a compound of the formula II
    especially of the formula IIA
    wherein Prot is a protecting group, Y is as defined for a compound of the formula I and X*, A1*, R2*, R3*, R5*, R6*, and R7* correspond to X, A1, R2, R3, R5, R6, and R7 in formula I, respectively, but with the proviso that reactive functional groups on these moieties (such as amino, imino, hydroxy, carboxy, sulfhydryl, amidino, guanidino, O-phosphono (—O—P(═O)(OH)2) are present in protected form at least if they could participate in undesired side reactions, to result in a compound of the formula III,
    especially of the formula IIIA,
    wherein X*, A1*, R2*, R3*, R5*, R6*, and R7* have the meanings just defined,    reacting the free hydroxyl group under oxidizing conditions to form a compound of the formula IV
    especially IVA,
    and removing remaining protecting groups to yield a compound of the formula I, or a salt thereof,    and, if desired, converting a free compound of the formula I, or especially IA, into a salt, a salt of a compound of the formula I into a different salt of a compound of the formula I, or especially IA, or into the free compound of the formula I, or especially IA, and/or converting a dehydrate analogue and/or five ring analogue of a compound of the formula I, or especially IA, into the corresponding compound of the formula I, or especially IA.
Suitable oxidizing conditions for the oxidation of a compound of the formula III or especially IIIA are usually using IBX in DMSO (J. Org. Chem. 1995, 60, 7272-7276); Pyridinium dichromate or Pyridinium chlorochromate (Tetrahedron Lett. 1979, 5, 399-402); oxalyl chloride, dimethyl sulfoxide and a tertiary amine (J. Peptide Sci. 2006, 12, 140-146), oxoammonium salts (J. Org. Chem. 1985, 50, 1332-1334); alkali hypochlorites catalyzed by oxoammonium salts (J. Org. Chem. 1989, 54, 2970-2972); oxoaminium salts (Tetrahedron Lett. 1988, 29, 5671-5672), RuCl2(PPh3)3 (Tetrahedron Lett. 1981, 22, 1605-1608); TEMPO (1 mol %) in the presence of sodium hypochlorite (Tetrahedron Lett. 1990, 31, 2177-2180); NaIO4, TEMPO, NaBr (Tetrahedron 2006, 62, 8928-8932); SiO2 supported vanadium(IV)oxide and t-BuOOH (Advanced Synthesis & Catalysis 2007, 349, 846-848). Preferably, the reaction is performed with IBX in DMSO or preferably in an inert solvent, such as tetrahydrofuran, in the presence of DMSO, at a temperature between 0-50° C., preferably between 20-25° C.    (ii/a) A further embodiment of the invention refers to the method or process described above, in addition comprising manufacturing the compound of the formula IV or especially IVA by a combination of Solid Phase Peptide Synthesis (especially For synthesis of the precursor XX or especially XXA given below for the oligopeptide precursor of the formula VIII or especially VIIIA given below, or of the oligopeptide precursor of the formula XXIV or especially XXIVA given below for the oligopeptide precursor of the formula XXV or especially XXVA given below) and Solution Phase synthesis (especially from the compounds just mentioned to the final product) from the corresponding starting amino acids and side chain precursors.    (iii/a) Yet a further embodiment of the invention relates to a method or process as described above, further comprising, for the synthesis of a compound of the formula II above, reacting a compound of the formula VI,
    especially VIA,
    wherein Prot is a protecting group, Y is as defined for a compound of the formula I and R2*, R3*, R5*, R6*, and R7* are as defined for a compound of the formula II above, with an acid of the formula VII
    or a reactive derivative thereof,    wherein X** is an amino protecting group or is X*, and wherein X* and A1* are as defined for a compound of the formula II above; and, if X** is an amino protecting group, removing said amino protecting group X** to yield the derivative of formula II (especially IIA) wherein, instead of X*, H (a hydrogen) is present and coupling the resulting amino group with an acyl group X* using the corresponding acid X*—OH wherein X* is as defined for a compound of the formula II defined above, or a reactive derivative thereof.    (iv/a) Another embodiment of the invention relates to the methods or processes described above, especially in the preceding paragraph, further comprising cyclization under lactamization of a linear, that is, not yet cyclic, precursor peptide of the compound of the formula VI, carrying an N-terminal amino group and a C-terminal carboxy group, under reaction conditions that allow for the formation of an amide bond from said amino and said carboxy group, preferably using Solution Phase chemistry.
Lactamizations in solutions are usually carried out at very low concentrations of the substrate in order to avoid oligomerizations and polymerizations. This requires huge amounts of solvents and very large reactors to carry out the reactions. For example, the macrolactamization of an oligopeptide is performed at a concentration of 2 mMols/liter in reference Yokokawa et al., Tetrahedron 2005, 61, 1459-1480. This difficulty can be circumvented by dissolving the tertiary base and the coupling reagent and, in a controlled way adding a solution of the oligopeptide to this solution. The controlled, especially slow, addition of the oligopeptide-solution generates permanently low concentrations of the activated oligopeptide in solution and thus prevents oligomerization and polymerization. The addition rate of the oligopeptide solution can be adjusted according to the reaction rate for the macrocyclization: if the macrocyclization is a fast reaction, the solution of the oligopeptide can be added fast. If the macrocyclization is slow, the addition of the solution must be slow to ensure permanent low concentration of the activated oligopeptide. Thus the controlled addition of the oligopeptide enables to work with much less solvent amounts and still maintaining the concentration of the activated oligopeptide below 10−3 mM, e.g. in the range from 10−4 to 10−6 mM or even lower. This variant of controlled addition of the oligopeptide to the coupling reagent solution is an embodiment of the invention.    (v/a) In yet a further embodiment, the invention relates to the method or process as described above, especially in the preceding paragraph, where the linear (this term where used meaning not yet cyclic) precursor peptide is of the formula VIII,
    especially VIIIA
    wherein Prot* is a protecting group that can be cleaved off selectively without affecting other protecting groups present and is stable during deprotection steps during synthesis of the linear precursor peptide (e.g. allyloxycarbonyl) and R2*, R3*, R5*, R6*, and R7* are as defined for a compound of the formula VI as described above, further comprising, after cyclisation of the compound of the formula VIII, especially VIIIA, removing the protecting group Prot* in situ to yield the compound of the formula VI, especially VIA.    (vi/a) In another embodiment, the invention relates to the method or process described above, especially in the preceding paragraph, where the linear precursor peptide of the formula VIII, especially VIIIA, is synthesized from the corresponding amino acids by solid phase peptide synthesis and subsequent cleavage from the employed solid support.    (vii/a) An embodiment of the invention further relates to the method or process as described above (especially in the preceding paragraph (vi/a)), further comprising either in a variant a), coupling an amino acid of the formula IX,
    especially of the formula IXA,
    wherein R3* is as defined for a compound of the formula II above and Prot** is an amino protecting group that can be removed on the resin without cleaving other bonds, or a reactive derivative of said amino acid, via an oxygen to a cleavable linker L which is bound to a solid resin RES (e.g. by reaction with a resin of the formula (X−L)z-RES wherein L and RES are as just defined, X is e.g. halo, e.g. chloro, and z is a number larger than zero, e.g. a natural number)    and removing the protecting group Prot**;    coupling the obtainable resin bound amino acid symbolized by the formula X,
    especially XA,
    in which RES and R3* are as defined for a compound of the formula IX, n is a (e.g. natural) number larger than zero and L is a cleavable linker, with an amino acid of the formula XI,
    especially XIA,
    wherein Prot* is as defined for a compound of the formula VIII above and R2* is as defined for a compound of the formula II above, or a reactive derivative of said amino acid, coupling the obtainable resin bound dipeptide symbolized by the formula XII,
    especially XIIA;
    in which Prot* is as defined for a compound of the formula VIII above, R2* and R3* are as defined for a compound of the formula II above, and n, L and RES are as defined for a compound of the formula X, via the free hydroxy group with an amino acid of the formula XIII,
    especially XIIIA,
    wherein Prot** is as defined for a compound of the formula IX and R7* is as defined for a compound of the formula II above, or a reactive derivative of said amino acid, and removing the protecting group Prot**;    or, in a variant b), coupling a dipeptide of the formula XXVII,
    especially of the formula XXVIIA,
    wherein R3* and Prot** are as described for a compound of the formula IX, especially IXA, and Prot* is as defined for a compound of the formula VIII above, or a reactive derivative of said dipeptide, to an amino acyl moiety, bound via an oxygen to a cleavable linker L which is bound to a solid resin RES, having the formula X,
    especially XA,
    that can be obtained as described under variant a), in which RES, and R3* are as defined for a compound of the formula IX and L and RES are as just defined;    and removing the protecting group Prot**;    and, after the reactions of variant a) or of variant b),    (viii/a) coupling the obtainable compound of the formula XIV,
    especially XIVA,
    wherein R2*, R3* and R7* are as defined for a compound of the formula II above, Prot* is as defined for a compound of the formula VIII above and n, L and RES are as defined for a compound of the formula X, with an amino acid of the formula XV,
    especially the formula XVA,
    in which R6* and Y are as defined for a compound of the formula II above and Prot** is as defined for a compound of the formula IX above, or a reactive derivative of said amino acid, and removing the protecting group Prot**;    (ix/a) preferably coupling the obtainable compound of the formula XVI
    especially the formula XVIA,
    wherein Y, R2*, R3*, R7* and R6* are as defined for a compound of the formula II above, Prot* is as defined for a compound of the formula VIII above and n, L and RES are as defined for a compound of the formula X, with an amino acid of the formula XVII,
    especially formula XVIIA,
    wherein R5* is as defined for a compound of the formula II above and Prot** is as defined for a compound of the formula IX, or a reactive derivative of said amino acid, and removing the protecting group Prot**,    and preferably    (x/a) finally coupling the resulting compound of the formula XVIII,
    especially XVIIIA,
    wherein Y, R2*, R3*, R7*, R6* and R5* are as defined for a compound of the formula II above, Prot* is as defined for a compound of the formula VIII above and n, L and RES are as defined for a compound of the formula X above, to an unnatural amino acid (=synthon) of the formula XIX,
    especially the formula XIXA,
    wherein Prot is as defined for a compound of the formula II above and Prot** is as defined for a compound of the formula IX, or an activated derivative of said synthon, and removing the protecting group Prot** to yield a compound of the formula XX,
    especially XXA,
    wherein Prot, Y, R2*, R3*, R7*, R6* and R5* are as defined for a compound of the formula II above, Prot* is as defined for a compound of the formula VIII above and n, L and RES are as defined for a compound of the formula X,    and    (xi/a) cleaving the solid phase bound peptide in formula XX off the solid phase L-RES to yield the corresponding compound of the formula VIII, especially VIIIA, as shown above.
Another embodiment of the invention relates to the synthesis of a compound of the formula II as given above according to section (i/a), preferably preceded by the reaction according to section (ii/a) or more preferably according to section (iii/a); preferably preceded by the reaction according to section (iv/a) or preferably (via), preferably preceded by the reaction according to section (vi/a), preferably preceded by the reaction according to section (x/a), preferably preceded by the reaction according to section (ix/a), preferably preceded by the reaction according to section (viii/a), preferably preceded by the reaction according to section (vii/a).    (i/b) Another embodiment of the invention relates to a method or process above, comprising, for the synthesis of the compound of the formula II given above, cyclization under lactamization of a linear, not yet cyclic, precursor peptide of the compound of the formula II, carrying an N-terminal amino group and a C-terminal carboxy group, under reaction conditions that allow for the formation of an amide bond from said amino and said carboxy group, preferably using Solution Phase chemistry.    (ii/b) A further embodiment of the invention relates to the method or process according to the preceding paragraph (i/b), where the linear precursor peptide is of the formula XXV,
    especially XXVA,
    wherein X*, A1*, R2*, R3*, R5*, R6*, R7* and Prot are as defined for a compound of the formula II above.    (iii/b) Another embodiment refers to the method or process according to the preceding paragraph (ii/b), further comprising, for the synthesis of the compound of the formula XXV, cleaving a compound of the formula XXIV,
    especially XXIVA,
    wherein X*, A1*, R2*, R3*, R5*, R6*, R7* and Prot are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, n is a natural number and Prot** is an amino protecting group that can be removed without parallel removal of the protecting group Prot and with the product remaining on the resin, and (before the cleavage, in parallel or subsequently to it) removing the protecting group Prot** to yield the compound of the formula XXV.    (iv/b) A further embodiment of the invention relates to the method or process according to the preceding paragraph (iii/b), further comprising, for the synthesis of the compound of the formula XXIV, coupling an amino acid of the formula XIX,
    especially XIXA,
    wherein Prot is as defined for a compound of the formula II above and Prot** is as defined for a compound of the formula XXIV above, or an activated derivative of said amino acid, with a compound of the formula XXIII,
    especially XXIIIA,
    wherein X*, A1*, R2*, R3*, R5*, R6* and R7* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number.    (v/b) Yet a further embodiment of the invention relates to the method or process according to the preceding paragraph (iv/b), further comprising, for the synthesis of the compound of the formula XXIII, coupling an amino acid of the formula XVII*
    especially XVIIA*,
    wherein R5* is as defined for a compound of the formula II above and Prot*** is an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin, or a reactive derivative of said amino acid, with a compound of the formula XXII,
    especially XXIIA
    wherein X*, A1*, R2*, R3*, R6* and R7* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number, and removing the protecting group Prot***.    (vi/b) In yet a further embodiment, the invention relates to the method or process according to the preceding paragraph (v/b), further comprising, for the synthesis of the compound of the formula XXII, coupling an amino acid of the formula XV*,
    especially XVA*
    in which R6* and Y are as defined for a compound of the formula II above and Prot*** is an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin, or a reactive derivative of said amino acid, with a compound of the formula XXI,
    especially XXIA,
    wherein X*, A1*, R2*, R3* and R7* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number, and removing the protecting group Prot***.    (vii/b) Another embodiment of the invention relates to the method or process according to the preceding paragraph (vi/b), further comprising, for the synthesis of a compound of the formula XXI, reacting an amino acid of the formula XIII*,
    especially XIIIA*,
    wherein Prot*** is an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin, and R7* is as defined for a compound of the formula II above, or a reactive derivative of said amino acid,    with the hydroxyl group of a compound of the formula XXVI,
    especially XXVIA,
    wherein X*, A1*, R2* and R3* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number;    and removing the protecting group Prot***.    (viii/b) In a further embodiment, the invention relates to the method or process according to the preceding paragraph (vii/b), further comprising, for the synthesis of a compound of the formula XXVI, especially XXVIA, coupling a resin bound dipeptide symbolized by the formula XII*,
    especially XIIA*
    in which Prot**** is a protecting group that can be cleaved off selectively without affecting other protecting groups present in a compound of the formula II as defined above and with the product remaining on the resin, R2* and R3* are as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number, after removal of the protecting group Prot**** via the thus obtainable free amino group, with. an acid of the formula VII
    wherein X** is an amino protecting group or is X*, and wherein X* and A1* are as defined for a compound of the formula II above, or a reactive derivative of said acid;    and, if X** is an amino protecting group, removing said amino protecting group X** to yield the derivative of formula II wherein, instead of X*, H is present and coupling the resulting amino group with an acyl group X* using the corresponding acid X*—OH wherein X* is as defined for a compound of the formula II above, or a reactive derivative of said acid.    (ix/b) A yet further embodiment of the invention relates to the method or process according to the preceding paragraph (viii/b), further comprising, for the synthesis of a compound of the formula XII, coupling a resin bound amino acid symbolized by the formula X,
    especially XA,
    wherein R3* is as defined for a compound of the formula II above, L is a cleavable linker, RES is a solid resin, and n is a natural number,    with an amino acid of the formula XI*,
    especially XIA*,
    wherein Prot**** is a protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin, and R2* is as defined for a compound of the formula II above, or a reactive derivative of said amino acid.    (x/b) A further embodiment of the invention relates to the method or process according to the preceding paragraph (ix/b), further comprising, for obtaining the resin bound amino acid of the formula X, coupling an amino acid of the formula IX*,
    especially IXA*,
    wherein R3* is as defined for a compound of the formula II in claim 1 and Prot*** is an amino protecting group can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin; or a reactive derivative of said amino acid of the formula IX, to a cleavable linker L which is bound to a solid resin RES, and removing the protecting group Prot***.    (i/c) Another embodiment of the invention relates to the method or process according to any one of the preceding paragraphs (i/a) to (x/b) where the symbols A1, R2, R3, R5, R6, R7, X and Y or the corresponding unprotected or protected moieties R2*, R3*, R5*, R6*, R7*, X* and Y3 are selected so that, in the resulting compound of the formula I, or a salt thereof,    A1 is the bivalent radical of L-glutamine bound via the carbonyl of its α-carboxy group to the amino group at the right of A1 in formula I and via its α-amino group to X, or is 2S-(2-hydroxy-3-phosphonooxy)-propionyl;    R2 is methyl;    R3 is isopropyl, isobutyl (2-methyl-n-propyl wherever used) or benzyl, especially isobutyl;    R5 is sec-butyl or benzyl, especially sec-butyl;    R6 is 4-hydroxybenzyl;    R7 is isopropyl or sec-butyl (1-methyl-n-propyl wherever used), especially sec-butyl;    X is acetyl or isobutyryl, or is absent if A1 is 2S-(2-hydroxy-3-phosphonooxy)-propionyl and    Y is methyl. This paragraph is also named paragraph a) below.    (i/d) In another particular embodiment, the invention relates to a method or process for converting a dehydrate of a compound of the formula I given above or in particular with the substituents as defined in the preceding paragraph (i/c) into the corresponding compound of the formula I, where the dehydrate has the formula V,
    especially VA,
    in which Y, X, A1, R2, R3, R5, R6 and R7 are as defined for a compound of the formula I above;    or especially a method or process for shifting the equilibrium of a mixture of a compound of the formula I and its corresponding dehydrate, and/or its corresponding hemiaminal analogue with a five-ring instead of the ahp structure in formula I which may also be formed as byproduct and has the formula V*,
    especially the formula VA*,
    in which Y, X, A1, R2, R3, R5, R6 and R7 are as defined for a compound of the formula I above, respectively;    in favor of the compound of the formula I,    said method or process comprising using an aqueous acid as reactive solvent to drive the reaction. This method can be used independently (e.g. also for the product of fermentation or biosynthesis) or in addition to the other processes or methods described above and below to increase the yield or to re-convert a compound of the formula V, especially VA, and/or the analogue with a five-membered ring instead of the ahp structure in formula I, into the corresponding compound of the formula I.
The method described for the conversion of the dehydrate and/or the five ring analogue (always regarding the desired ahp ring) into the desired compound of the formula I or especially IA, e.g. of Compound A-dehydrate from Example 3 B into Compound A, enables a straight-forward synthesis of this class of compounds. Up to now, an acidic treatment as final step had to be circumvented in order to avoid the dehydration of the product.    (i/e) A further embodiment of the invention relates to the method according to the preceding paragraph (i/d), wherein the acid is a carboxylic acid, especially a halo substituted C1-8alkanoic acid, more especially trifluoroacetic acid or trichloroacetic acid.    (i/f) The invention, in yet a further embodiment, relates to a compound of the formula II,
    wherein Prot is a protecting group, Y is as defined for a compound of the formula I in the first instance above or in particular as or claim 17 and X*, A1*, R2*, R3*, R5*, R6*, and R7* correspond to X, A1, R2, R3, R5, R6, and R7 in formula I as defined in claim 1 or in paragraph (ia) given above, respectively, however with the proviso that reactive functional groups on these moieties are present in protected form.    (i/g) In a further embodiment, the invention relates to a novel compound selected from the group consisting of compounds of the formula II, III, IV, V, VI, VIII, X, XII, XIV, XVI, XVIII, XIX, XX, XXI, XXII, XXIII, XXIV, XXV, XXVI and XXVIII, and especially of the formula IIA, IIIA, IVA, VA, VIA, VIIIA, XA, XIIA, XIVA, XVIA, XVIIIA, XIXA, XXA, XXIA, XXIIA, XXIIIA, XXIVA, XXVA, XXVIA and XXVIIIA, yet more especially to the group consisting of the following compounds given in the examples: From Scheme 1: compound 2, compound 3, compound 4, synthon 1; from Scheme 2: compound 5; Fmoc-Leu-Linker-Resin according to Example 1B(2); Fmoc-Thr-Leu-Linker-Resin according to Example 1B(3); Fmoc-Gln(Trt)-Thr-Leu-Linker-Resin according to Example 1B(4); Isobutyryl-Gln(Trt)-Thr-Leu-Linker-Resin according to Example 1B(5); Isobutyryl-Gln(Trt)-Thr(Ile-Fmoc)-Leu-Linker-Resin according to Example 1B(6); the product of Example 1B(7)=Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Fmoc)-Leu-Linker-Resin (previously named: Isobutyryl-Gln(Trt)-Thr(Ile-N-me-Tyr(tBu)-Fmoc)-Leu-Linker-Resin); Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Fmoc)-Leu-Linker-Resin (previously named: Isobutyryl-Gln(Trt)-Thr(Ile-N-me-Tyr(tBu)-Ile-Fmoc)-Leu-Linker-Resin) according to Example 1B(8); Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon 1-H)-Leu-Linker-Resin (previously named: Isobutyryl-Gln(Trt)-Thr(Ile-N-Me-Tyr(tBu)-Ile-Synthon 1-H)-Leu-Linker-Resin) according to example 1B(9). Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon 1-H)-Leu-OH (previously named: Isobutyryl-Gln(Trt)-Thr(Ile-N-Me-Tyr(tBu)-Ile-Synthon 1-H)-Leu-OH) according to example 1B(10) and Scheme 3; H-Thr-Leu-Resin according to example 1B(12); H-Gln(Trt)-Thr-Leu-Resin according to example 1B(13); Isobutyryl-Gln(Trt)-Thr-Leu-Resin according to example 1B(14); Isobutyryl-Gln(Trt)-Thr(Ile-H)-Leu-Resin according to example 1B(15); Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-H)-Leu-Resin according to example 1B(16); Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-H)-Leu-Resin according to example 1B(17); Isobutyryl-Gln(Trt)-Thr(Ile-Tyr(tBu)Me-Ile-Synthon 1-H)-Leu-Resin according to example 1B(18); from Scheme 3, Compound 6 and/or 7 (the latter preferred); from Scheme 4: Compound 8 and the 5-ring hemiaminal-isomer; from Scheme 5, precursor peptide 2, compound 9, compound 10 (this one being preferred in the present enumeration regarding Scheme 5), and/or compound 11; Fmoc-Thr-Leu-Trt-Tentagel-S from Example 2A(1); Fmoc-Gln(Trt)-Thr-Leu-Trt-Tentagel-S according to Example 2A(2); Ac-Gln(Trt)-Thr-Leu-Trt-Tentagel-S according to Example 2A(3); Ac-Gln(Trt)-Thr(Val-Fmoc)-Leu-Trt-Tentagel-S according to Example 2A(4); Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Fmoc)-Leu-Trt-Tentagel-S (previously named: Ac-Gln(Trt)-Thr(Val-N-Me-Tyr(tBu)-Fmoc)-Leu-Trt-Tentagel-S) according to Example 2A(5); Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Fmoc)-Leu-Trt-Tentagel-S (previously named: Ac-Gln(Trt)-Thr(Val-N-Me-Tyr(tBu)-Phe-Fmoc)-Leu-Trt-Tentagel-S) according to Example 2A(6); and Ac-Gln(Trt)-Thr(Val-Tyr(tBu)Me-Phe-Synthon1-H)-Leu-OH (previously named: Ac-Gln(Trt)-Thr(Val-N-Me-Tyr(tBu)-Phe-Synthon1-H)-Leu-OH) (precursor peptide 2) according to Example 2A(7).
The following definitions (or also definitions already included above) can replace more general terms used in invention embodiments above and below in order to define further embodiments of the invention, with either one, two or more or all general terms being replaceable by the more specific terms in order to define such invention embodiments:
A bivalent moiety of an amino acid with a terminal carboxy or carbamoyl group is preferably an alpha-carbamoyl or carboxyl-C1-8-substituted amino acid, especially the bivalent moiety of asparagine or glutamine, and is bound at its right hand side in formula I via a carbonyl (preferably the carbonyl of its α-carboxyl group) to the rest of the molecule.
C1-8-alkanoyl or phosphorylated hydroxy-C1-8-alkanoyl (C1-8-alkanoyl carrying both a hydroxyl and a phosphono (—O—P(═O)(OH)2) group) A1 is e.g. 2,3-dihydroxy-propanoyl (preferably in S-form) or 2-hydroxy-3-phosphono-propanoyl (preferably in S-form).
R2 and R2* are C1-8-alkyl, especially methyl wherever mentioned.
R3 is the side chain of an amino acid, especially of a natural amino acid. Preferably, it is C1-8 alkyl which may be branched or linear. Most especially, C1-8alkyl is n-(2-methyl)propyl(isobutyl), n-(1-methylpropyl(sec-butyl) or methyl, that is, the amino acid carrying the moiety is leucine, isoleucine or valine.
R3* is the corresponding side chain in protected form if a functional group is present that has to be hindered to participate in a reaction. Preferably, it is C1-8alkyl which may be branched or linear, especially as defined in the preceding paragraph.
A “side chain of an amino acid” may be selected from any moiety, e.g. a mono- or polycyclic, linear, saturated, unsaturated (e.g. with conjugated double bonds) or partially saturated organic moiety, e.g. with up to 20 carbon atoms and 0 to 5 heteroatoms in the basis structure independently selected from N, O and S replacing the corresponding number of carbon atoms, and may be substituted by up to three moieties selected from amino, imino, hydroxy, carboxy, carbamoyl, sulfhydryl, amidino, guanidino, O-phosphono (—O—P(═O)(OH)2). Preferably, the side chains are selected from those of the 20 standard alpha-amino acids arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, tyrosine, valine and further proline (then with internal cyclization including the alpha-amino group).
For the amino acids, either their names or the customary three letter codes are used in the present disclosure, in accordance with the following table:
Amino acidThree letter codeAlanineAlaArginineArgAsparagineAsnAspartic acidAspAsparagine or aspartic acidAsxCysteineCysGlutamic acidGluGlutamineGlnGlutamine or glutamic acidGlxGlycineGlyHistidineHisisoleucineIleLeucineLeuLysineLysMethionineMetPhenylalaninePheProlineProSerineSerThreonineThrTryptophanTryTyrosineTyrValineVal
R5 is the side chain of an amino acid, preferably a standard amino acid. Preferably, it is C1-8 alkyl which may be branched or linear and which is unsubstituted or substituted by phenyl. Most especially it is benzyl, n-(2-methyl)propyl, isobutyl or methyl, that is, the amino acid carrying the moiety is phenylalanine, leucine, isoleucine or valine.
R6 is the side chain of a hydroxy amino acid, especially of tyrosine.
R7 is the side chain of an amino acid, especially of a natural amino acid. Preferably, it is C1-8alkyl which may be branched or linear. Most especially it is n-(2-methyl)propyl(isobutyl), n-(1-methyl)propyl(sec-butyl) or methyl, that is, the amino acid carrying the moiety is leucine, isoleucine or valine.
C1-8-alkyl can be linear or branched one or more times; for example, it can be n-(2-methyl)propyl, n-(1-methyl)propyl or methyl.
All of the compounds can, where salt-forming groups such as basic groups, e.g. amino or imino, or acidic groups, e.g. carboxyl or phenolic hydroxyl, are present, be used in free form or as salts or as mixtures of salts and free forms. Thus where ever a compound is mentioned, this includes all these variants. For example, basic groups may form salts with acids, such as hydrohalic acids, e.g. HCl, sulfuric acid or organic acids, such as acetic acid, while acidic groups may form salts with positive ions, e.g. ammonium, alkylammonium, alkali or alkaline-earth metal salt cations, e.g. Ca, Mg, Na, K or Li cations, or the like.
“Or the like” or “and the like”, wherever used in this disclosure, refers to the fact that other alternatives to those mentioned preceding such expression are known to the person skilled in the art and may be added to those expressions specifically mentioned; in other embodiments, “or the like” and “and the like” may be deleted in one or more or all invention embodiments.
The protecting groups Prot, Prot*, Prot**, Prot***, Prot**** and any further protecting groups present on the moieties A*, R2*, R3*, R5*, R6*, R7*, X*, where ever mentioned throughout the present description and claims, are selected so that they allow for orthogonal protection.
Orthogonal protection is a strategy allowing the deprotection of multiple protective groups one (or more but not all) at the time where desired each with a dedicated set of reaction conditions without affecting the other protecting group(s) or bonds to resins, e.g. via linkers on solid synthesis resins. In other terms: The strategy uses different classes of protecting groups that are removed by different chemical mechanisms, also using appropriate linkers in the case of solid phase peptide synthesis (where the linker-resin bond might together be considered as a carboxy protecting group).
Preferably, the protecting groups are selected as follows:
The protecting group Prot is preferably selected so as to resist removal of any other protecting groups used or present during the synthesis according to the invention of a depsipeptide, e.g. able to resist mild bases (see Prot*), but removable with fluoride ion (especially under anhydrous conditions), e.g. Bu4N+F− (also if created in situ, e.g. using Bu4N+Cl− with KF.H2O, KF with 18-crown-6, LiBr with 18-crown-6, BF3.diethylether, pyridine-HF, HF in urea, Et3N(HF)3 (wherein Et is ethyl) or the like, where the solvent is e.g. selected from the group consisting of N,N-dimethylformamide, acetonitrile, chloroform and tetrahydrofurane.
Preferably, Prot is an ether protecting group, especially selected from the group consisting of silyl protecting groups in which the silyl moiety carries up to three organic moieties bound via a carbon (optionally via a further Si atom), such as tert-butyldiphenylsilyl, trimethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, triphenylsilyl, diphenylmethylsilyl, ti-tert-butyldimethylsilyl, tert-butylmethoxyphenylsilyl, tris(trimethylsilyl)silyl or the like.
Prot* is a protecting group that can be cleaved off selectively without affecting other protecting groups present, and also without affecting the depsipeptide forming ester bond or a linker to a resin RES, and is stable during deprotection steps during synthesis of the linear precursor peptide (e.g. removal of allyloxycarbonyl); it is preferably a protecting group removable by specific triphenylphosphin complexes in the presence of metal hydrides or other reductants, e.g. (PH3P)4Pd preferably in combination with di-n-butyl tin hydride or tri-n-butyl tin hydride, phenylsilane, sodium borohydride or dimedone, in an appropriate solvent, e.g. tetrahydrofurane, and is preferably not cleavable under conditions that allow for the removal of a protecting group Prot**; for example, and Prot* is selected from the group consisting of C3-C8alk-2-enyloxycarbonyl moieties, e.g. allyloxycarbonyl (Alloc), 1-isopropylallyloxycarbonyl, 4-nitrocinnamyloxycarbonyl and 3-(3′-pyridyl)prop-2-enyloxycarbonyl.
Prot** is a protecting group that can be removed on the resin without cleaving other bonds (no cleavage of an amino acid or peptide bound via the carbonyl of its (especially α-carboxyl group to the binding via a linker L mentioned below; also without cleaving off the protecting group Prot once present), especially a protecting group removable without cleavage of an ester (instead of an amide) bond in a depsipeptide or depsipeptide precursor and under conditions other than those for the protecting groups Prot* and Prot, while preserving the binding via the linker to a resin RES where present; it is preferably removable by a mild base e.g. piperidine, morpholine, dicyclohexylamine, p-dimethyl-amino-pyridine, diisopropylamine, piperazine, tris-(2-aminoethyl)amine in an appropriate solvent, e.g. N,N-dimethylformamide, methylene chloride; Prot** is, e.g., selected from the group consisting of fluoren-9-ylmethoxycarbonyl (Fmoc); 2-(2′ or 4′-pyridyl)ethoxycarbonyl and 2,2-bis(4′nitrophenyl)ethoxycarbonyl.
Prot*** (an amino protecting group that can be cleaved off selectively without affecting other protecting groups present and with the product remaining on the resin) and Prot**** (a protecting group that can be cleaved off selectively without affecting other protecting groups present in a compound of the formula II as defined above and below and with the product remaining on the resin) are preferably protecting groups that can be removed as Prot** and are, e.g., selected from those mentioned for R**, e.g. fluoren-9-ylmethoxycarbonyl (Fmoc).
The preferred orthogonal synthesis method in this case makes use of the Fmoc method known in general for peptide synthesis using solid phase peptide synthesis combined with solution phase macrolactamization and further chemical conversions.
Alternatively, e.g. the Boc protecting group might be used instead of Fmoc Prot**, Prot*** and Prot****.
However, this will require different side chain protecting groups and also the hydroxy-group of N-methyl-Tyr will then have to be protected in a different way to maintain orthogonality of the protection groups.
Other protecting groups present as well as the binding linker to a resin RES where present are preferably not removable under conditions under which Prot* and Prot** can be removed, e.g. in A*, the amide can be N-protected e.g. with trityl (triphenylmethyl) (cleavage e.g. with trifluoro acetic acid (TFA); in R6* a tyrosine hydroxy can be protected as t-butyl-ether, or protected by tert-butyldimethylsilyl, methoxymethyl, Boc (tert-butoxycarbonyl) or arylacetate (cleavage with TFA).
Appropriate protecting groups are known in the art, as well methods for their introduction and removal. For example, the protecting groups, their introduction and removal methods may be selected from those described in standard textbooks such as “Protective Groups in Organic Synthesis”, 3rd ed., T. W. Green and P. G. M. Wuts (Eds.). J. Wiley & Sons, Inc., New York etc. 1999.
The protecting groups Prot, Prot*, Prot**, Prot***, Prot**** and other protecting groups are thus not limited to those mentioned above—rather they should fulfill conditions that make them appropriate for orthogonal protection, e.g. as described above or below.
It is recommended to avoid too basic conditions (though the bases described for Fmoc cleavage, such as piperidine, are usually allowable) to avoid cleavage of the depsipeptide (ester) bond.
Among the possible solid support for Solid Phase Peptide Synthesis (SPPS), the following may be mentioned:                Gel-type supports without or with spacer: These are highly solvated polymers with an equal distribution of functional groups. This type of support is the most common, and includes:            Polystyrene: Styrene cross-linked with e.g. 1-2% divinylbenzene; Polyacrylamide or polymethacrylamide: as hydrophilic alternative to polystyrene; Polyethylene glycol (PEG): PEG-Polystyrene (PEG-PS) is more stable than polystyrene and spaces the site of synthesis from the polymer backbone; PEG-based supports: Composed of a PEG-polypropylene glycol network or PEG with polyamide or polystyrene (these already include a spacer, PEG);            Surface-type supports: Materials developed for surface functionalization, including controlled pore glass, cellulose fibers, and highly cross-linked polystyrene.        Composites: Gel-type polymers supported by rigid matrices.        
Usually these gels carry reactive groups to which a linker L as mentioned for various precursors above and below can be bound. For example, such groups include aminomethyl groups, polyethyleneglycol groups with a terminal hydroxy, and the like.
Any such support can be used in the embodiments of the present invention.
Gel type supports are used in another special embodiment of the invention, Among these, polystyrene (divinylbenzene crosslinked); polyacrylamide and polymethacrylamide resins are especially preferred.
Among the possible linkers, all commonly known and appropriate may be used.
Examples in possible embodiments of the invention are the 2-methoxy-4-benzyloxy-benzyl alcohol linker (a Sasrin-Linker, Sasrin stands for superacid sensitive resin, binds the amino acids or peptides via alcoholic OH); the trityl linker family (e.g, Trityl, 2Cl-Trityl, which bind the amino acids or peptides via OH); the 4-(2,4-dimethoxyphenylhydroxymethyl)phenoxymethyl-Linker (Rink-Acid-Linker, binds the amino acids or peptides via OH); or tris(alkoxy)benzyl ester linkers (HAL-Linker, binds the amino acids or peptides via OH).
Where reactive derivatives of acids, especially amino acids, or peptides, e.g. dipeptides, are mentioned, they may be formed in situ or may be used as such.
Reactive (or active) derivatives used as such include the acyl-halides, e.g. acyl-chlorides, -fluorides or -nitrophenyl esters, e.g. the 2,4-dinitrophenyl esters, or acid anhydrides (symmetric or e.g. with acetic acid) of the carboxy groups of the acids to be reacted.
For in situ amino acid activation, customary coupling agents may be applied. Such reagents are known to the person skilled in the art and can be purchased conveniently from many sources, e.g. Aldrich ChemFiles—Peptide Synthesis (Aldrich Chemical Co., Inc., Sigma-Aldrich Corporation, Milwaukee, Wis., USA) Vol. 7 No. 2, 2007 (see http://www.sigmaaldrich.com/etc/medialib/docs/Aldrich/Brochure/al_chemfile_v7_n2.Par.0001.File.tmp/al_chemfile_v7_n2.pdf). Among the possible coupling agents for amide and ester bond synthesis the following may be mentioned:
Triazoles, uronium or hexafluorophosphonium derivatives, e.g. 1-hydroxy-benzotriazole (HOBt), 1-hydroxy-7-aza-benzotriazole (HOAt), ethyl 2-cyano-2-(hydroxyimino)acetate, 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium (HATU), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate (HBTU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluoroborate (TBTU), 2-succinimido-1,1,3,3-tetramethyluronium-tetrafluoroborate (TSTU), 2-(5-norbornen-2,3-dicarboximido)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), O-[(cyano(ethoxycarbonyl)-methyliden)amino]-1,1,3,3-tetramethyluronium-tetrafluoroborate (TOTU), O-(benzotriazol-1-yl)-1,3-dimethyl-1,3-dimethylene uronium hexafluorophosphate (HBMDU), O-(benzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate (HBPyU), O-(benzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uronium hexafluorophosphate (HBPipU), 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt), 1-hydroxy-7-aza-benzotriazole and its corresponding uronium or phosphonium salts, designated HAPyU and AOP, 1-cyano-2-ethoxy-2-oxoethylideneaminooxy-dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), chlorotripyrrolidinophosphonium hexafluorophosphate (PyCloP), or the like;
Carbodiimides, e.g. dicyclohexylcarbodiimide, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, 1-tert-butyl-3-ethylcarbodiimide, N-cyclohexyl-N′-2-morpholinoethyl)carbodiimide or diisopropylcarbodiimide (especially for ester formation via O-acyl urea formation of the carboxylic group); or    active ester forming agents, e.g. 2-mercaptobenzothiazole (2-MBT),    azide forming agents, e.g. diphenyl phosphoryl azide,    acid anhydrides, such as propane phosphonic acid anhydride,    acid halogenation agents, e.g. 1-chloro-N,N,2-trimethyl-1-propenylamine, chloro-N,N,N′,N′-bis(tetramethylene)formamidinium tetrafluoroborate or hexafluorophosphate, chloro-N,N,N′,N′-tetramethlformamidinium hexafluorophosphate, fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate, fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate,    or the like, or mixtures of two or more such agents.
Also for the ester coupling of compounds of the formula XII or XIIA with those of the formula XIII or XIIIA, respectively, or of compounds of the formula XIII* or XIIIA*with those of the formula XXVI or XXVIA, respectively, the corresponding reactive carboxyl compounds can be used or formed in situ. Here, especially MSNT is preferred as coupling agent as this allows for the maintenance of high stereospecificity.
The reaction may, where appropriate, be conducted in the presence of a mild base (e.g. N-methylmorpholine, a trialkylamine, e.g. ethyldiisopropylamine, a di-(alkyl)aminopyridine, such as N,N-dimethylaminopyridine, or the like (taking care that the conditions are not so basic as to allow for the hydrolysis of ester groups, e.g. the depsipeptide ester group, present in precursors of the compound of the formula I), where appropriate or required in the presence of an appropriate solvent or solvent mixture, e.g. an N,N dialkyl-formamide, such as dimethylformamide, a halogenated hydrocarbon, e.g. dichloromethane, N-alkylpyrrolidones, such as N-methylpyrrolidone, nitriles, e.g. acetonitrile, or further an aromatic hydrocarbon, e.g. toluene, or mixtures of two or more, where, provided an excess of coupling agent is present, also water may be present. The temperatures may be ambient temperature or lower or higher, e.g. in the range from −20° C. to 50° C.
The amino acids of the formula IX, IXA, XI, XIA, XIII, XIIIA, XV, XVA, XVII, XVIIA, XXVII (obtainable e.g. by Solution Phase synthesis), XVII*, XVIIA*, XV*, XVA*, XIII*, XIIIA*, XI*, XIA*, IX* and XIA* are known or they can be synthesized according to methods known in the art, they are commercially available, and/or they can be synthesized in analogy to methods known in the art.
Also the remaining starting materials, e.g. the acid of the formula XIX or VII, or the dipeptide of the formula XXVII or XXVIIA, are known or they can be synthesized according to methods known in the art, they are commercially available, and/or they can be synthesized in analogy to methods known in the art.
For example, the synthon of the formula XIX can be prepared as described in Example 1 A(4) (which is a specific embodiment of the invention) or in analogy thereto. The synthesis of the intermediate compound 1 (Scheme 1) is described in Tetrahedron 61, 1459-1480 (2005).
The coupling reactions for dipeptides make use of the corresponding carboxylic groups of amino acids in free form or in activated form.